Briefly, plasma was supplemented with 2% (w/w) of human fibrinogen conjugated to Alexa Fluor 488 (Thermo Scientific). Clot formation was initiated with the addition of 1U thrombin (EMD Millipore) in the presence of 2.4 mM calcium as described31 (link).
For fibrin-vimentin interaction, plasma was supplemented with 2% (w/w) of human fibrinogen conjugated to Alexa Fluor 647 (Thermo Scientific) and with mouse anti-human Vimentin (V9, Sigma-Aldrich) or mouse IgG (isotype control) prior to initiating clot formation with the addition of 1U thrombin (EMD Millipore) in the presence of 2.4 mM calcium as described31 (link). Fibrin clots were washed three times with phosphate buffred saline (PBS) and incubated immediately with a secondary anti-mouse IgG-CF488A (1:1000, Sigma) conjugate for 1 hr. Then, fibrin-clot were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min and washed three times for visualization by confocal microscopy.
For fibrin-vimentin interaction, plasma was supplemented with 2% (w/w) of human fibrinogen conjugated to Alexa Fluor 647 (Thermo Scientific) and with mouse anti-human Vimentin (V9, Sigma-Aldrich) or mouse IgG (isotype control) prior to initiating clot formation with the addition of 1U thrombin (EMD Millipore) in the presence of 2.4 mM calcium as described31 (link). Fibrin clots were washed three times with phosphate buffred saline (PBS) and incubated immediately with a secondary anti-mouse IgG-CF488A (1:1000, Sigma) conjugate for 1 hr. Then, fibrin-clot were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min and washed three times for visualization by confocal microscopy.